Center for Cellular Immunotherapies, Abramson Cancer Center and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address: aposey@mail.med.upenn.edu.

2

Center for Cellular Immunotherapies, Abramson Cancer Center and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Department of Pathology, The University of Chicago, Chicago, IL 60637, USA.

5

Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

6

Department of Pathology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

7

Center for Cellular Immunotherapies, Abramson Cancer Center and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Pathology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

8

Center for Cellular Immunotherapies, Abramson Cancer Center and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Pathology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. Electronic address: cjune@exchange.upenn.edu.

Abstract

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.

5E5 mAb and 5E5 CAR Specifically Recognize the Tn-MUC1, but Not the Peptide Alone

(A) ELISA-peptide assay of glycopeptides OSM, aOSM, Muc1-9Tn, and unconjugated Muc1-60-mer with antibodies 3C9, 5F4, 3F1, and 5E5. The peptides were coated in the plate wells at concentrations of 2,000 ng/mL in bicarbonate buffer and halved in dilutions until 1 ng/mL. The bottom right panel used starting concentration of 2,000 ng/mL and diluted 8-fold until 233 fg/mL (noted with **). Statistical significance is calculated using an unpaired t test comparing 5F4 ELISA for Muc1-9Tn and Muc1-60-mer, 3F1 ELISA for OSM and aOSM, and 5E5 ELISA for Muc1-9Tn and Muc1-60-mer. p < 0.0001.(B) Graphical representation of the chimeric antigen receptor developed using the 5E5 scFv, CD8α hinge, and transmembrane domain, 4-1BB and CD3zeta endodomain.(C) Indirect ELISA assays quantifying the cytokine production in supernatant from 5E5 CAR and CD19 CAR T cells cultured on peptide-bound plates for 24 hr. Peptides were plated starting at concentrations of 2,000 ng/mL and diluted 256-fold until 1.82 ag/mL. 10 μg/mL of OKT3 mAb was used as a control T cell stimulant. Statistical significance of 5E5 CAR T cells on Muc1-9Tn versus Muc1-60-mer peptide is p = 0.0101 for IL-2 secretion and p = 0.0012 for IFN-γ secretion.

5E5 and CD19 CAR T cells were tested in a chromium release lysis assays at effector:target ratios of 1:1 to 30:1. MC813-70 CAR T cells known to exhibit normal tissue toxicity were used as a positive control. In addition the various CAR T cells were tested on wild-type Jurkat and Jurkat with reconstituted COSMC. Statistical comparisons are between 5E5 CAR and CD19 CAR in the positive controls Jurkat E6-1 and Jurkat 19COSMC. All other comparisons are made between MC813-70 and 5E5. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.